Mutant of enzyme, mutant of glycosyltransferase and application of mutant in preparation of salidroside

Abstract

The invention relates to the field of bioengineering, in particular to a mutant of enzyme, a mutant of glycosyltransferase and application of the mutant in preparation of salidroside. The invention provides a mutant of enzyme, which has the following amino acid site mutation on the basis of wild type glycosyl transferase: the 308th site is mutated from I to Q, the 333rd site is mutated from V to R, the 363rd site is mutated from W to S and the 386th site is mutated from S to G, and the amino acid sequence of the wild type glycosyl transferase is as shown in SEQ ID NO: 1. Glycosyltransferase and sucrose synthase with improved thermal stability are obtained through structural rational design and deep learning modification, tyrosol can be glycosylated to generate salidroside in a mode of regenerating ADP-glucose, the price of ADP is only about 20% of that of UDP, and the method has the cost advantage.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, in particular to an enzyme mutant, a glycosyltransferase mutant and applications thereof in the preparation of salidroside. Background Art

[0002] Rhodiola rosea, a perennial herb found on the plateau, has medicinal properties, including its dried roots and rhizomes, known for invigorating qi and blood circulation, relieving asthma, and promoting blood circulation. Its main active ingredients are salidroside and tyrosol. Along with cordyceps sinensis, saffron, and snow lotus, Rhodiola rosea is considered one of the four most precious Tibetan medicinal herbs and has been used in pharmaceuticals, health supplements, and skincare products in recent years. Representative species of the Rhodiola genus include Rhodiola crenulata, Rhodiola kirilowii, Rhodiola sachalinensis, and Rhodiola rosea, among over 90 others. Due to overharvesting and environmental degradation, wild resources are becoming increasingly depleted. Although Rhodiola rosea has begun to be cultivated artificially in the Gannan region, the long growing period and high costs have prevented large-scale cultivation and failed to meet market demand.

[0003] Thanks to the gene discovery of salidroside synthesizing glycosyltransferase in plants, the Tianjin Institute of Engineering and Tianjin University carried out research on the fermentation synthesis of salidroside using Escherichia coli and Saccharomyces cerevisiae as chassis cells. However, due to the low activity of glycosyltransferase and insufficient supply of UDP-glucose in cells, the conversion of tyrosol to salidroside was incomplete, which limited its industrial application. Summary of the Invention

[0004] In light of this, the present invention provides enzyme mutants, glycosyltransferase mutants, and their use in the preparation of salidroside. Through rational structural design and deep learning-based transformation, the present invention produces glycosyltransferases and sucrose synthases with enhanced thermal stability. These enzymes can glycosylate tyrosol to produce salidroside by regenerating ADP-glucose. The price of ADP is only approximately 20% of that of UDP, offering a cost advantage.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides an enzyme mutant, which, based on a wild-type glycosyltransferase, has the following amino acid site mutations: site 308 is mutated from I to Q, site 333 is mutated from V to R, site 363 is mutated from W to S, and site 386 is mutated from S to G. The amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO: 1.

[0006] In some embodiments of the present invention, the amino acid sequence of the mutant is shown in SEQ ID NO:5.

[0007] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the wild-type glycosyltransferase is shown in SEQ ID NO: 2.

[0008] The present invention also provides a mutant of glycosyltransferase, which, based on the above mutant, has the following amino acid site mutations: position 21 mutates from A to I, position 83 mutates from M to N, and position 407 mutates from Q to P.

[0009] In some embodiments of the present invention, the amino acid sequence of the mutant of the glycosyltransferase is shown in SEQ ID NO:7.

[0010] The present invention also provides an enzyme composition comprising: a mutant of the above enzyme and / or a mutant of the above glycosyltransferase, and sucrose synthase.

[0011] In some embodiments of the present invention, in the enzyme composition, the amino acid sequence of the sucrose synthase is shown in SEQ ID NO: 3.

[0012] The present invention also provides nucleic acid molecules encoding mutants of the above enzymes, mutants of the above glycosyltransferases and / or the above enzyme composition.

[0013] In some embodiments of the present invention, the nucleic acid molecule comprises: The nucleotide sequence of the nucleic acid molecule encoding the mutant of the enzyme is shown in SEQ ID NO: 6; and / or The nucleotide sequence of the nucleic acid molecule encoding the mutant of the glycosyltransferase is shown in SEQ ID NO: 8; and / or The nucleotide sequence of the nucleic acid molecule encoding the sucrose synthase is shown in SEQ ID NO: 4.

[0014] The present invention also provides a recombinant vector comprising: the above nucleic acid molecule and acceptable gene elements.

[0015] The present invention also provides a host for transforming and / or transfecting the above recombinant vector.

[0016] The present invention also provides a product comprising: a mutant of the above enzyme, a mutant of the above glycosyltransferase, the above enzyme composition, the above nucleic acid molecule, the above recombinant vector and / or the above host, and acceptable adjuvants or excipients.

[0017] The present invention also provides the use of the mutant of the above enzyme, the mutant of the above glycosyltransferase, the above enzyme composition, the above nucleic acid molecule, the above recombinant vector, the above host and / or the above product in the preparation of salidroside.

[0018] The present invention also provides a method for preparing salidroside, wherein the raw materials are converted into salidroside by any of the following methods: (a) a mutant of the above enzyme; or (b) a mutant of the above-mentioned glycosyltransferase; or (c), the enzyme composition described above; or (d), the nucleic acid molecule described above; or (e) the above-mentioned recombinant vector; or (f) the above hosts; or (g) the above products; The raw materials include sucrose, tyrosol and ADP.

[0019] In some embodiments of the present invention, in the above preparation method, the temperature during the conversion is 30-60°C.

[0020] In some embodiments of the present invention, in the above preparation method, when a mutant of the above enzyme is used, the temperature during the conversion is 30°C.

[0021] In some embodiments of the present invention, in the above preparation method, when the mutant of the above glycosyltransferase is used, the temperature during the conversion is 60°C.

[0022] In some embodiments of the present invention, in the above preparation method, the enzyme activity of the mutant of the enzyme is 13.3 U / mL; the enzyme activity of the mutant of the glycosyltransferase is 80.3 U / mL; and the enzyme activity of the sucrose synthase is 20.6~83.8 U / mL.

[0023] In some embodiments of the present invention, in the above preparation method, the final concentration of sucrose is 204.5-409 mM; the final concentration of tyrosol is 202.7-405.3 mM; and the final concentration of ADP is 0.2 mM.

[0024] The beneficial effects of the present invention include: (1) The modified UDP glycosyltransferase can use cheap ADP as a substrate instead of expensive UDP; (2) The secondary modified ADP glycosyltransferase can react at a high temperature of 60°C, which greatly shortens the reaction time compared to 30°C, and can complete reactions with high substrate concentrations above 400 mM. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0026] Figure 1 Shown are the electrophoresis results of AtUGT85A1 and AcSUS proteins expressed in shake flasks; Figure 2 The predicted structure of AtUGT85A1 is aligned with the crystal structure of the SrUGT76G1-UDP-RA complex, with UDP shown as a ball-and-stick model. Figure 3 The residues of SrUGT76G1 that interact with UDP are shown (the rectangular boxes are marked as the corresponding residues of AtUGT85A1); Figure 4 The residues Ser307, Ile308, Val333, Trp363, and Ser386 that may interact with UDP in the predicted structure of AtUGT85A1 are shown; Figure 5 Local sequence alignment of AtUGT85A1 residues that may interact with UDP is shown; Figure 6 Shown is the global single-point mutation Pythia energy heat map of AtUGT85A1; Figure 7 Shown is the high performance liquid chromatography (HPLC) spectrum of pure salidroside; Figure 8 Shows the mass spectrum of pure salidroside. DETAILED DESCRIPTION

[0027] The invention discloses an enzyme mutant, a glycosyltransferase mutant and applications of the mutants in preparing salidroside.

[0028] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0029] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0030] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0031] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0032] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0033] The amino acid sequence of the UDP-glycosyltransferase AtUGT85A1 from Arabidopsis thaliana is: MGSQIIHNSQKPHVVCVPYPAQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDMDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASA IILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSITVLSVKQLVEFAWGLAGSGKEFLWVIRPDLVAGEEAMVPPDFLMETKDRSMLA SWCPQEKVLSHPAIGGFLTHCGWNSILESLSCGVPMVCWPFFADQQMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD (SEQ ID NO:1); Amino acid sequence of sucrose synthase AcSUS derived from Acidithiobacillus caldus: MIEALRQQLLDDPRSWYAFLRHLVASQRDSWLYTDLQRACADFREQLPEGYAEGIGPLEDFVAHTQEVIFRDPWMVFAWRPRPGRWIYVRIHREQLALEELSTDAYLQAKEGIVGLGAEGEAVLTVDFRDFRPVSRRLRDESTIGDGLTHLNRRLAGRIFSDLAAGRSQILEFLSLHRLDGQNLMLSNGNTDFDSLRQTVQYLGTLPRETPWAEIREDMRRRGFAPGWGNTAGRVRETMRLLMDLLDSPSPAALESFLDRIPMISRILIVSIHGWFAQDKVLGRPDTGGQVVYILDQARALEREMRNRLRQQGVDVEPRILIATRLIPESDGTTCDQRLEPVVGAENVQILRVPFRYPDGRIHPHWISRFKIWPWLERYAQDLEREVLAELGSRPDLIIGNYSDGNLVATLLSERLGVTQCNIAHALEKSKYLYSDLHWRDHEQDHHFACQFTADLIAMNAADIIVTSTYQEIAGNDREIGQYEGHQDYTLPGLYRVENGIDVFDSKFNIVSPGADPRFYFSYARTEERPSFLEPEIESLLFGREPGADRRGVLEDRQKPLLLSMARMDRIKNLSGLAELYGRSSRLRGLANLVIIGGHVDVGNSRDAEEREEIRRMHEIMDHYQLDGQLRWVGALLDKTVAGELYRVVADGRGVFVQPALFEAFGLTVIEAMSSGLPVFATRFGGPLEIIEDGVSGFHIDPNDHEATAERLADFLEAARERPKYWLEISDAALARVAERYTWERYAERLMTIARIFGFWRFVLDRESQVMERYLQMFRHLQWRPLAHAVPME (SEQ ID NO:3); Amino acid sequence of the engineered ADP - glycosyltransferase AtUGT85A1 - I308Q / V333R / W363S / S386G (AGT): MGSQIIHNSQKPHVVCVPYPAQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDMDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSQTVLSVKQLVEFAWGLAGSGKEFLWRIRPDLVAGEEAMVPPDFLMETKDRSMLASSCPQEKVLSHPAIGGFLTHCGWNGILESLSCGVPMVCWPFFADQQMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD (SEQ ID NO:5); Amino acid sequence of the modified ADP-ribosyltransferase AGT-A21I / M83N / Q407P: MGSQIIHNSQKPHVVCVPYPIQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDNDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSQTVLSVKQLVEFAWGLAGSGKEFLWRIRPDLVAGEEAMVPPDFLMETKDRSMLASSCPQEKVLSHPAIGGFLTHCGWNGILESLSCGVPMVCWPFFADQPMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD (SEQ ID NO:7);

[0034] In Comparative Example 1 and Examples 1 to 5 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0035] The present invention will be further described below in conjunction with the embodiments: Comparative Example 1 Synthesis of Salidroside by UDP Glycosyltransferase and Sucrose Synthase The UDP glycosyltransferase AtUGT85A1 from Arabidopsis thaliana and the sucrose synthase AcSUS from Acidithiobacillus caldus were selected and codon optimized for the host Escherichia coli using the Invitrogen GeneArt online tool GeneOptimizer. The corresponding optimized nucleotide sequences were synthesized and constructed in the pET-22b vector.

[0036] The resulting plasmids pET22b-atUGT85A1 and pET22b-acSUS were transformed into BL21 (DE3) competent cells and expanded to 2000 mL of LB liquid medium (Amp). Expression was induced at 30°C with 0.1 mM IPTG. Bacteria were harvested by cold centrifugation and resuspended in a disruption buffer (100 mM K2HPO4∙3H2O, 10 mM KH2PO4, 200 mM NaCl, pH 7.6) at a ratio of 1 g of wet bacteria. The cells were then ultrasonically disrupted to obtain a crude enzyme solution. Protein electrophoresis was used to detect expression. Figure 1 ), AtUGT85A1 and AcSUS were mainly expressed in the supernatant.

[0037] Weigh 35 g of sucrose (204.5 mM, reaction volume 500 mL), 14 g of tyrosol (202.7 mM), and 0.04 g of UDP (0.2 mM) and dissolve them in 400 mL of pure water. Adjust the pH to 6.0 and dilute to 450 mL. After preheating to 30°C, add 30 mL of crude AtUGT85A1 enzyme solution (12.5 U / mL) and 20 mL of crude AcSUS enzyme solution (20.6 U / mL) to start the reaction.

[0038] During the reaction, 100 μL of the sample was diluted with 900 μL of methanol. Another 100 μL sample was then diluted 100-fold with 900 μL of methanol. Tyrosol and salidroside were then analyzed by high-performance liquid chromatography (HPLC). As shown in Table 1, the conversion rate reached 86.85% after 48 h of reaction, but this reaction took a long time.

[0039] Table 1

[0040]

[0041] Example 1 Glycoside-selective modification of UDP glycosyltransferase The glycosyltransferase SrUGT76G1-UDP-RA complex structure (PDB: 6INI) from Stevia was used as a reference model, and the AlphaFold predicted AtUGT85A1 structure was aligned with it as a whole ( Figure 2 ), the skeleton RMSD 1.006Å indicates that the structures are relatively similar, especially the UDP binding region. LigPlot is used to display the residues that interact with UDP ( Figure 3 ), Val309 and Trp338 of SrUGT76G1 constrain the uracil ring of UDP, and Ser283 and Ser361 interact with the diphosphate of UDP and may be involved in glycosyl transfer. Sequence and structure alignment ( Figure 4 and Figure 5 ) Shows the residues Ser307 (Ser283), Ile308 (Thr284), Val333 (Val309), Trp363 (Trp338), and Ser386 (Ser361) that may interact with AtUGT85A1 and UDP, with the corresponding SrUGT76G1 residues in brackets.

[0042] To accommodate a larger adenine ring, Val333 and Trp363 were first selected as modification sites for AtUGT85A1. Meanwhile, Ser307, Ile308, and Ser386 were fine-tuned to accommodate the shift of diphosphate. Primers listed in Table 2 were designed and the mutant plasmids were constructed using the QuickChange Site-Directed Mutagenesis Kit (Agilent).

[0043] Table 2

[0044]

[0045] The resulting plasmids were transformed into BL21(DE3) competent cells and expanded to 200 mL of LB liquid medium (Amp). Expression was induced at 30°C with 0.1 mM IPTG. Bacteria were harvested by cold centrifugation and resuspended in 9 mL of disruption buffer at a ratio of 1 g of wet bacteria. The resulting enzyme was then disrupted by ultrasonication to obtain the corresponding crude enzyme solution.

[0046] 1 mL of crude enzyme solution was pipetted into a final volume of 10 mL of reaction solution (10 mM tyrosol, 5 mM ADP-glucose, pH 6.0). The reaction was incubated at 30°C, 300 rpm, and 5 mL of acetonitrile was added to terminate the reaction. The supernatant was centrifuged and analyzed for salidroside by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results are shown in Table 3, indicating that V333R was the optimal single-point mutation.

[0047] Table 3

[0048]

[0049] In Table 3, * Definition of product concentration compared to wild type: "-" less than 1.5 times; "+" 1.5-2.5 times; "++" 2.5-3.5 times; "+++" more than 3.5 times.

[0050] Based on the AtUGT85A1-V333R mutant, other mutation sites were superimposed and constructed, cultured, expressed, and fragmented in the same manner as described above. The results are shown in Table 4, with AtUGT85A1-I308Q / V333R / W363S / S386G being the optimal mutation.

[0051] Table 4

[0052]

[0053] In Table 3, * Definition of product concentration compared to the AtUGT85A1-V333R mutant: "+" less than 1.5 times; "++" 1.5-2 times; "+++" more than 2 times.

[0054] Example 2 Synthesis of Salidroside by Glycosyltransferase Mutants and Sucrose Synthase AtUGT85A1-I308Q / V333R / W363S / S386G mutant BL21 (DE3) glycerol culture was expanded to 2000 mL of LB liquid medium (Amp) and expression was induced at 30°C with 0.1 mM IPTG. Cells were harvested by cold centrifugation and resuspended in 4 mL of disruption buffer (100 mM K₂HPO₄∙3H₂O, 10 mM KH₂PO₄, 200 mM NaCl, pH 7.6) at a ratio of 1 g wet cells. The cells were then sonicated to obtain a crude enzyme solution.

[0055] Weigh 70 g of sucrose (204.5 mM, reaction volume 1000 mL) and 28 g of tyrosol (202.7 mM) and dissolve them in 800 mL of pure water. Adjust the pH to 6.0 and make the volume to 900 mL. Divide the mixture into two portions and dissolve 0.04 g of ADP (0.2 mM) and 0.04 g of UDP (0.2 mM) in each portion. Preheat to 30°C and add 30 mL of AtUGT85A1-I308Q / V333R / W363S / S386G crude enzyme solution (13.3 U / mL) and 20 mL of AcSUS crude enzyme solution (20.6 U / mL) to start the reaction.

[0056] During the reaction, 100 μL of the sample was diluted with 900 μL of methanol, and then 100 μL of the sample was taken and diluted 100-fold with 900 μL of methanol before being analyzed by high-performance liquid chromatography (HPLC) for tyrosol and salidroside. As shown in Table 5, the ADP group had a conversion rate of 87.37% after 46 hours of reaction, comparable to the efficiency of the AtUGT85A1 and AcSUS enzymes in Comparative Example 1 in regenerating UDG-glucose to salidroside. However, the UDP group had a conversion rate of only 38.01% after 46 hours of reaction, indicating that the modified AtUGT85A1-I308Q / V333R / W363S / S386G is an ADP-glucose-selective glycosyltransferase, designated "AGT."

[0057] Table 5

[0058]

[0059] Example 3 Thermal stability modification of ADP glycosyltransferase AGT The AtUGT85A1 structure predicted by AlphaFold was uploaded to Pythia, a protein mutation prediction tool based on structure self-supervised learning, to generate a global single-point mutation energy heat map ( Figure 6 ), the top 20 single-point mutations with the lowest ΔΔG values ​​are shown in Table 6, and their structures were analyzed.

[0060] Table 6

[0061]

[0062] The surface sites M83, V310, Q407, L467, M472 and the sites A21 and G151 that affect loop flexibility were selected, and the primers listed in Table 7 were designed using the nucleotide sequence of AGT (SEQ ID NO. 6) as a template to construct mutant plasmids using the QuickChange site-directed mutagenesis kit (Agilent).

[0063] Table 7

[0064]

[0065] In Table 7, * Definition of product concentration compared to AGT: "-" less than 1.5 times; "+" 1.5-3 times; "++" 3-6 times; "+++" more than 6 times.

[0066] The resulting plasmids were transformed into BL21(DE3) competent cells and expanded to 200 mL of LB liquid medium (Amp). Expression was induced at 30°C with 0.1 mM IPTG. Bacteria were harvested by cold centrifugation and resuspended in 9 mL of disruption buffer at a ratio of 1 g of wet bacteria. The resulting enzyme was then disrupted by ultrasonication to obtain the corresponding crude enzyme solution.

[0067] 1 mL of crude enzyme solution was pipetted into a final volume of 10 mL of reaction solution (10 mM tyrosol, 5 mM ADP-glucose, pH 6.0). The reaction was incubated at 60°C, 300 rpm, and 5 mL of acetonitrile was added to terminate the reaction. The supernatant was centrifuged and analyzed for salidroside by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results are shown in Table 7, indicating that Q407P was the optimal single-point mutation.

[0068] Based on the AGT-Q407P mutant, other mutation sites were superimposed and constructed, cultured, expressed, and fragmented in the same manner as described above. The results are shown in Table 8, with AGT-A21I / M83N / Q407P being the optimal mutation.

[0069] Table 8

[0070]

[0071] In Table 8, * Definition of product concentration compared to the AGT-Q407P mutant: "-" less than 1-fold; "+" 1-2-fold; "++" 2-3-fold; "+++" more than 3-fold.

[0072] Example 4 Synthesis of Salidroside by ADP-glycosyltransferase and Sucrose Synthase A BL21(DE3) glycerol culture of the AGT-A21I / M83N / Q407P mutant was expanded to 2000 mL of LB liquid medium (Amp) and induced for expression at 30°C with 0.1 mM IPTG. Cells were harvested by cold centrifugation and resuspended in 4 mL of disruption buffer (100 mM K₂HPO₄∙3H₂O, 10 mM KH₂PO₄, 200 mM NaCl, pH 7.6) at a ratio of 1 g of wet cells. The cells were then sonicated to obtain a crude enzyme solution.

[0073] Weigh 35 g of sucrose (204.5 mM, reaction volume 500 mL), 14 g of tyrosol (202.7 mM), and 0.04 g of ADP (0.2 mM) and dissolve them in 400 mL of pure water. Adjust the pH to 6.0 and dilute to 480 mL. After preheating to 60°C, add 10 mL of AGT-A21I / M83N / Q407P crude enzyme solution (80.3 U / mL) and 10 mL of AcSUS crude enzyme solution (83.8 U / mL) to start the reaction.

[0074] During the reaction, 100 μL of the sample was diluted with 900 μL of methanol. Another 100 μL sample was then diluted 100-fold with 900 μL of methanol before being analyzed by high-performance liquid chromatography (HPLC) for tyrosol and salidroside. As shown in Table 9, the conversion rate was 88.72% after 22 hours of reaction. Compared with the AGT and AcSUS enzyme reactions in Example 2, the reaction time was significantly shortened and the enzyme dosage was lower.

[0075] Table 9

[0076]

[0077] Example 5 Synthesis of high-concentration salidroside by ADP glycosyltransferase and sucrose synthase Weigh 70 g of sucrose (409.0 mM, reaction volume 500 mL), 28 g of tyrosol (405.3 mM), and 0.04 g of ADP (0.2 mM) and dissolve them in 400 mL of pure water. Adjust the pH to 6.0 and dilute to 480 mL. After preheating to 60°C, add 15 mL of AGT-A21I / M83N / Q407P crude enzyme solution (80.3 U / mL) and 10 mL of AcSUS crude enzyme solution (83.8 U / mL) to start the reaction.

[0078] During the reaction, 100 μL of the sample was diluted with 900 μL of methanol. Another 100 μL sample was then diluted 100-fold with 900 μL of methanol before being analyzed by high-performance liquid chromatography (HPLC) for tyrosol and salidroside. As shown in Table 10, after 22 hours of reaction, the conversion rate was 83.84%, and the salidroside concentration of 102 g / L was the highest reported.

[0079] After the reaction solution was passed through a ceramic membrane to remove proteins, it was purified using a C18 preparative liquid phase with a mobile phase of 30% methanol. The target peak was collected by rotary evaporation and dried under constant temperature in a vacuum to obtain a crude product. The crude product was dissolved by heating with 5 volumes of methanol / ethyl formate (1:1, v / v) at 50°C, then cooled to 4°C for crystallization, and dried under constant temperature in a vacuum to obtain a pure product. The purity was 99.98% (HPLC) Figure 7 ), by mass spectrometry ( Figure 8 ) characterized as salidroside.

[0080] Table 10

[0081]

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An enzyme mutant, characterized in that Based on the wild-type glycosyltransferase, the following amino acid site mutations are present: site 308 mutates from I to Q, site 333 mutates from V to R, site 363 mutates from W to S, and site 386 mutates from S to G. The amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO:

1.

2. The mutant of the enzyme according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

5.

3. A mutant of a glycosyltransferase, characterized in that Based on the mutant as described in claim 1 or 2, the mutant has the following amino acid position mutations: position 21 mutates from A to I, position 83 mutates from M to N, and position 407 mutates from Q to P.

4. The mutant of glycosyltransferase according to claim 3, characterized in that Its amino acid sequence is shown in SEQ ID NO:

7.

5. An enzyme composition, characterized in that include: A mutant of the enzyme according to claim 1 or 2 and / or a mutant of the glycosyltransferase according to claim 3 or 4, and sucrose synthase.

6. A nucleic acid molecule encoding a mutant of the enzyme according to claim 1 or 2, a mutant of the glycosyltransferase according to claim 3 or 4 and / or an enzyme composition according to claim 5.

7. The nucleic acid molecule according to claim 6, wherein include: The nucleotide sequence of the nucleic acid molecule encoding the mutant of the enzyme is shown in SEQ ID NO: 6; and / or The nucleotide sequence of the nucleic acid molecule encoding the mutant of the glycosyltransferase is shown in SEQ ID NO: 8; and / or The nucleotide sequence of the nucleic acid molecule encoding the sucrose synthase is shown in SEQ ID NO:

4.

8. A recombinant vector, characterized in that include: The nucleic acid molecule according to claim 6 or 7 and an acceptable genetic element.

9. A host, characterized in that Transform and / or transfect the recombinant vector according to claim 8.

10. The product is characterized in that include: The mutant of the enzyme according to claim 1 or 2, the mutant of the glycosyltransferase according to claim 3 or 4, the enzyme composition according to claim 5, the nucleic acid molecule according to claim 6 or 7, the recombinant vector according to claim 8 and / or the host according to claim 9, and acceptable adjuvants or excipients.

11. Use of the mutant of the enzyme according to claim 1 or 2, the mutant of the glycosyltransferase according to claim 3 or 4, the enzyme composition according to claim 5, the nucleic acid molecule according to claim 6 or 7, the recombinant vector according to claim 8, the host according to claim 9 and / or the product according to claim 10 in the preparation of salidroside.

12. A method for preparing salidroside, characterized in that: The raw materials are converted into salidroside by any of the following methods: (a) a mutant of the enzyme according to claim 1 or 2; or (b) a mutant of the glycosyltransferase according to claim 3 or 4; or (c) an enzyme composition as claimed in claim 5; or (d) a nucleic acid molecule according to claim 6 or 7; or (e) the recombinant vector according to claim 8; or (f) the host according to claim 9; or (g) The product according to claim 10; The raw materials include: sucrose, tyrosol and ADP.

13. The preparation method according to claim 12, wherein The temperature during the conversion is 30-60°C.

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